Compositions and methods for the biosynthesis of vanillin or vanillin beta-D glucoside

ABSTRACT

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to have reduced levels or activity of one or more alcohol dehydrogenases or aldehyde reductase thereby increasing the production of vanillin or vanillin beta-D-glucoside.

INTRODUCTION

This application is a continuation of U.S. application Ser. No.14/905,228 filed Jan. 14, 2016, now U.S. Pat. No. 10,000,782, which isthe National Stage of international Application. No. PCT/US2014/046315filed Jul. 11, 2014, which claims benefit of priority to U.S.Provisional Application Ser. No. 61/846,658, filed Jul. 16, 2013, theteachings of which are incorporated herein by reference in theirentireties.

BACKGROUND

Vanillin is one of the world's most important flavor compounds with aglobal market of 180 million dollars. Natural vanillin is derived fromthe cured seed pods of the vanilla orchid (Vanilla planifolia), but mostof the world's vanillin is synthesized from petrochemicals or wood pulplignins. Production of natural vanillin from the vanilla pod is alaborious and slow process, which requires hand pollination of theflowers and a 1-6 month curing process of the harvested green vanillapods (Ramachandra & Ravishankar (2000) J. Sci. Food Agric. 80:289-304).Production of 1 kilogram (kg) of vanillin requires approximately 500 kgof vanilla pods, corresponding to pollination of approximately 40,000flowers. Today only about 0.25% (40 tons out of 16,000) of vanillin soldannually originates from vanilla pods, while most of the remainder issynthesized chemically from lignin or fossil hydrocarbons, in particularguaiacol. Synthetically produced vanillin is sold for approximately $15per kg, compared to prices of $1200-4000 per kg for natural vanillin(Walton, et al. (2003) Phytochemistry 63:505-515).

SUMMARY OF THE INVENTION

This invention provides a recombinant host cell having the followingcharacteristics: the recombinant host cell produces vanillin and/orvanillin beta-D-glucoside; and the recombinant host cell has reducedproduction or activity of a first alcohol dehydrogenase and reducedproduction of one or more second alcohol dehydrogenases, one or morealdehyde reductases, or a combination thereof. In some embodiments, thefirst alcohol dehydrogenase is Alcohol Dehydrogenase 6 (ADH6). Inanother embodiment, the one or more second alcohol dehydrogenasesinclude Alcohol Dehydrogenase 7 (ADH7), Genes de Respuesta a Estres 2(GRE2), or an ortholog thereof. In a further embodiment, the aldehydereductase includes Aldehyde Reductase Intermediate 1 (ARI1), AldehydeReductase YGL039W, or an ortholog thereof. In certain embodiments, therecombinant host cell further includes a nucleic acid encoding an AROMpolypeptide, a nucleic acid encoding a catechol-O-methyltransferase(COMT) polypeptide, a nucleic acid encoding a 3-dehydroshikimatedehydratase (3DSD) polypeptide, a nucleic acid encoding an aromaticcarboxylic acid reductase (ACAR) polypeptide, a nucleic acid encoding aphosphopantetheine transferase (PPTase) polypeptide, a nucleic acidencoding an uridine 5′-diphosphoglucosyl transferase (UGT) polypeptideand/or a nucleic acid encoding a vanillyl alcohol oxidase (VAO).Microorganism such as Saccharomyces cerevisiae, Schizosaccharomycespombe or Escherichia coli are provided as are plant or plant cells suchas Physcomitrella or tobacco.

A recombinant yeast cell; a vanillin and/or vanillin glucoside extractisolated from the recombinant host cell or recombinant yeast cell; aconsumable, e.g., a food product, pharmaceutical composition, a dietarysupplement, a nutraceutical, a dental hygienic composition, a tabletopsweetener, or a cosmetic product containing the extract; and a methodfor producing vanillin and/or vanillin beta-D-glucoside are alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of de novo biosynthesis of vanillin (4) andoutline of the different vanillin catabolites and metabolic sideproducts, i.e., dehydroshikimic acid (1), protocatechuic acid (2),protocatechuic aldehyde (3), vanillic acid (5), protocatechuic alcohol(6), vanillyl alcohol (7), and vanillin β-D-glucoside (8), found in anorganism expressing 3DSD, ACAR, OMT, and UGT and a phophopantheteinetransferase (PPTase). Open arrows show primary metabolic reactions inyeast; black arrows show enzyme reactions introduced by metabolicengineering; diagonally striped arrows show undesired inherent yeastmetabolic reactions.

FIG. 2 shows the production of vanillyl alcohol glucoside, vanillylalcohol, vanillin β-D-glucoside and vanillin in a strain lacking one ormore functional alcohol dehydrogenases.

FIG. 3 shows the production of vanillyl alcohol glucoside, vanillylalcohol, vanillin β-D-glucoside and vanillin in a strain lacking gre2(EFSC2055 gre2) or gre2 and aril (EFSC2055 gre2 aril) as compared to theparental strain EFSC2055. *, p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the discovery that knocking out certainalcohol dehydrogenases and/or aldehyde reductases, or similar enzymes,lowers the amount of vanillic alcohol that is formed as a byproduct. SeeFIGS. 1 and 2. This is of commercial importance because the presence ofalcohol creates inefficiencies in certain steps of the downstreampurification of vanillin or vanillin glucoside if the alcohol is allowedto accumulate.

Therefore, the present invention is a recombinant host that is capableof producing vanillin or vanillin glucoside, but fails to produce, orhas reduced production of, one or more alcohol dehydrogenases and/or oneor more aldehyde reductases. A recombinant host that produces or iscapable of producing vanillin or vanillin glucoside is a host cell thatexpresses the necessary biosynthetic enzymes to produce vanillin orvanillin glucoside from a primary substrate, e.g., glucose, or from anintermediate molecule, e.g., dehydroshikimic acid, protocatechuic acid,protocatechuic aldehyde, or vanillic acid. See FIG. 1.

A recombinant host that fails to produce an enzyme, has reducedproduction of an enzyme, or lacks a functional enzyme, includes anorganism that have been recombinantly modified such that the geneencoding the enzyme is knocked out, an organism with one or more pointmutations in the enzyme which reduces or diminishes enzyme activity, oran organism wherein the promoter of the gene encoding the enzyme hasbeen modified or removed so that the enzyme is not expressed orexpressed at a reduced level compared to a wild-type organism.

Many methods for genetic modification of target genes are known to oneskilled in the art and may be used to create the recombinant host ofthis invention. Modifications that may be used to reduce or eliminateexpression of a target enzyme are disruptions that include, but are notlimited to, deletion of the entire gene or a portion of the geneencoding an enzyme; inserting a DNA fragment into a gene encoding theenzyme (in either the promoter or coding region) so that the enzyme isnot expressed or expressed at lower levels; introducing a mutation intothe coding region for the enzyme, which adds a stop codon or frame shiftsuch that a functional enzyme is not expressed; and introducing one ormore mutations into the coding region of an enzyme to alter amino acidsso that a non-functional or a less enzymatically active enzyme isexpressed. In addition, expression of an enzyme may be blocked byexpression of an antisense RNA or an interfering RNA, and constructs maybe introduced that result in cosuppression. In addition, the synthesisor stability of the transcript may be lessened by mutation. Similarly,the efficiency by which an enzyme is translated from mRNA may bemodulated by mutation. All of these methods may be readily practiced byone skilled in the art making use of the known sequences encoding thealcohol dehydrogenases and/or aldehyde reductases of this invention.

Alcohol dehydrogenase and aldehyde reductase sequences from a variety oforganisms are well-known in the art and the selection of the targetgene(s) will be dependent upon the host selected. Representative alcoholdehydrogenase (ADH) and aldehyde reductase sequences, which may betargeted in accordance with the present invention are listed in Table 1.One skilled in the art may choose specific modification strategies toeliminate or lower the expression of an alcohol dehydrogenase and/oraldehyde reductase as desired to facilitate vanillin and/or vanillinglucoside production.

TABLE 1 Amino Acid Nucleotide Sequence Sequence SEQ SEQ Accession ID IDSource Target No. NO: Accession No. NO: S. cerevisiae Adh6 NP_014051 1NM_001182831 2 S. cerevisiae Adh7 NP_010030 3 NM_001178812 4 S.cerevisiae GRE2 NP_014490 5 NM_001183405 6 S. cerevisiae YDR541CNP_010830 7 NM_001180849 8 S. cerevisiae ARI1 NP_011358 9 NM_00118102210 S. cerevisiae YGL039W NP_011476 11 NM_001180904 12 S. pombeSPAC513.07 NP_593981 13 NM_001019407 14 Arabidopsis CAD3 NP_179780 15NM_127758 16 thaliana A. thaliana CAD9 NP_195643 17 NM_120093 18 A.thaliana CAD2 NP_179765 19 NM_127743 20 A. thaliana AT1G51410 NP_17555221 NM_104019 22 A. thaliana AT5G19440 NP_197445 23 NM_121949 24

In some embodiments, the recombinant host cells has reduced productionor activity of a first alcohol dehydrogenase and reduced production ofone or more second alcohol dehydrogenases, one or more aldehydereductases, or a combination thereof. In particular embodiments, thefirst alcohol dehydrogenase is ADH6 or an ortholog thereof, e.g., CAD9,CAD3 or CAD2 from A. thaliana. In another embodiment, the one or moresecond alcohol dehydrogenases are selected from the group of ADH7, GRE2(Genes de Respuesta a Estres 2), or an ortholog thereof, e.g., AT1G51410or AT5G19440; and the aldehyde reductase is selected from the group ofARI1 (Aldehyde Reductase Intermediate 1), Aldehyde Reductase YGL039W, oran ortholog thereof, e.g., SPAC513.07 or YDR541C).

DNA sequences surrounding one or more of the above-referenced sequenceare also useful in some modification procedures and are available foryeasts such as for Saccharomycse cerevisiae in the complete genomesequence coordinated by NCBI (National Center for BiotechnologyInformation) with identifying BioProject Nos. PRJNA1128, PRJNA13838,PRJNA43747, PRJNA48559, PRJNA52955, PRJNA48569, PRJNA39317. Additionalexamples of yeast genomic sequences include that of Schizosaccharomycespombe, which is included in BioProject Nos. PRJNA127, PRJNA13836, andPRJNA20755. Genomic sequences of plants are also known in the art andthe genomic sequence of Arabidopsis thaliana is included in BioProjectNos. PRJNA116, PRJNA10719, PRJNA13190, and PRJNA30811. Other genomicsequences can be readily found by one of skill in the art in publiclyavailable databases.

In particular, DNA sequences surrounding an alcohol dehydrogenase oraldehyde reductase coding sequence are useful for modification methodsusing homologous recombination. For example, sequences flanking the geneof interest are placed on either side of a selectable marker gene tomediate homologous recombination whereby the marker gene replaces thegene of interest. Also partial gene sequences and flanking sequencesbounding a selectable marker gene may be used to mediate homologousrecombination whereby the marker gene replaces a portion of the targetgene. In addition, the selectable marker may be bounded by site-specificrecombination sites, so that following expression of the correspondingsite-specific recombinase, the resistance gene is excised from the geneof interest without reactivating the latter. The site-specificrecombination leaves behind a recombination site which disruptsexpression of the alcohol dehydrogenase or aldehyde reductase. Thehomologous recombination vector may be constructed to also leave adeletion in the gene of interest following excision of the selectablemarker, as is well known to one skilled in the art.

Deletions may be made using mitotic recombination as described in Wach,et al. ((1994) Yeast 10:1793-1808). This method involves preparing a DNAfragment that contains a selectable marker between genomic regions thatmay be as' short as 20 bp, and which bound a target DNA sequence. ThisDNA fragment can be prepared by PCR amplification of the selectablemarker gene using as primers oligonucleotides that hybridize to the endsof the marker gene and that include the genomic regions that canrecombine with the yeast genome. The linear DNA fragment can beefficiently transformed into yeast and recombined into the genomeresulting in gene replacement including with deletion of the target DNAsequence.

Moreover, promoter replacement methods may be used to exchange theendogenous transcriptional control elements allowing another means tomodulate expression such as described in Mnaimneh, et al. ((2004) Cell118(1):31-44).

Hosts cells of use in this invention include any organism capable ofproducing vanillin and/or vanillin glucoside either naturally orsynthetically, e.g., by recombinant expression of one or more genes ofthe vanillin and/or vanillin glucoside biosynthetic pathway (FIG. 1). Anumber of prokaryotes and eukaryotes are suitable for use inconstructing the recombinant microorganisms described herein, e.g.,gram-negative bacteria, gram-positive bacteria, yeast or other fungi. Aspecies and strain selected for use as a vanillin and/or vanillinglucoside production strain is first analyzed to determine whichproduction genes are endogenous to the strain and which genes are notpresent. Genes for which an endogenous counterpart is not present in thestrain are assembled in one or more recombinant constructs, which arethen transformed into the strain in order to supply the missingfunction(s).

Exemplary prokaryotic and eukaryotic species are described in moredetail below. However, it will be appreciated that other species may besuitable. For example, suitable species may be in a genus Agaricus,Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia,Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia,Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces,Schizosaccharomyces, Sphaceloma, Xanthophyllomyces Yarrowia andLactobacillus. Exemplary species from such genera include Lentinustigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichiapastoris, Physcomitrella patens, Rhodoturula glutinis 32, Rhodoturulamucilaginosa, Phaffia rhodozyma UBV-AX, Xanthophyllomyces dendrorhous,Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis and Yarrowialipolytica. In some embodiments, a microorganism can be an Ascomycetesuch as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomycespombe, Aspergillus niger, or Saccharomyces cerevisiae. In someembodiments, a microorganism can be a prokaryote such as Escherichiacoli, Rhodobacter sphaeroides, or Rhodobacter capsulatus. It will beappreciated that certain microorganisms can be used to screen and testgenes of interest in a high throughput manner, while othermicroorganisms with desired productivity or growth characteristics canbe used for large-scale production of vanillin beta-D-glucoside.

Specific non-limiting examples of useful recombinant hosts are describedin WO 01/40491, as well as in Hansen et al. (2009) Appl. Environ.Microbiol. 75:2765-2774 and Brochado, et al. (2010) Microbial CellFactories 9:84, wherein the recombinant host according to this inventioncontains a heterologous nucleic acid encoding a mutant COMT polypeptideand/or mutant AROM polypeptide instead of the OMT genes described in WO01/40491.

One preferred recombinant host to use with the present invention is S.cerevisiae, which may be recombinantly engineered as described herein.S. cerevisiae is a widely used chassis organism in synthetic biology,and can be used as the recombinant microorganism platform. There arelibraries of mutants, plasmids, detailed computer models of metabolismand other information available for S. cerevisiae, allowing for rationaldesign of various modules to enhance product yield. Methods are knownfor making recombinant microorganisms. The VG4 strain of S. cerevisiae(Brochado, et al. (2010) Microb. Cell Fact. 9:84) is particularlyuseful. VG4 has the genotype of pdc1Δgdh1Δ↑GDH2.

Aspergillus species such as A. oryzae, A. niger and A. sojae are widelyused microorganisms in food production, and can also be used as therecombinant microorganism platform. Thus, the recombinant host may beAspergillus spp. Nucleotide sequences are available for genomes of A.nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, andA. terreus, allowing rational design and modification of endogenouspathways to enhance flux and increase product yield. Metabolic modelshave been developed for Aspergillus, as well as transcriptomic studiesand proteomics studies. A. niger is cultured for the industrialproduction of a number of food ingredients such as citric acid andgluconic acid, and thus species such as A. niger are generally suitablefor the production of food ingredients such as vanillin and vanillinglucoside.

E. coli, another widely used platform organism in synthetic biology, canalso be used as the recombinant microorganism platform. Thus, therecombinant host may be E. coli. Similar to Saccharomyces, there arelibraries of mutants, plasmids, detailed computer models of metabolismand other information available for E. coli, allowing for rationaldesign of various modules to enhance product yield. Methods similar tothose described above for Saccharomyces can be used to make recombinantE. coli microorganisms.

Rhodobacter can be used as the recombinant microorganism platform. Thus,the recombinant host may be Rhodobacter spp. Similar to E. coli, thereare libraries of mutants available as well as suitable plasmid vectors,allowing for rational design of various modules to enhance productyield. Methods similar to those described above for E. coli can be usedto make recombinant Rhodobacter microorganisms.

Physcomitrella mosses, when grown in suspension culture, havecharacteristics similar to yeast or other fungal cultures. This generais becoming an important type of cell for production of plant secondarymetabolites, which can be difficult to produce in other types of cells.Thus, the recombinant host may be a Physcomitrella spp.

In some embodiments, the recombinant host is a plant or plant cells thatincludes the one or more genes of the vanillin and/or vanillin glucosidebiosynthetic pathway. A plant or plant cell can be modified to expressthe vanillin and/or vanillin glucoside biosynthetic pathway with aconcurrent knockout of one or more alcohol dehydrogenases and/oraldehyde reductases. The plant or plant cells can be stably transformedto retain the introduced nucleic acid with each cell division. A plantor plant cell can also be transiently transformed such that theheterologous nucleic acid is not integrated into its genome. Transientlytransformed cells typically lose all or some portion of the introducednucleic acid with each cell division such that the introduced nucleicacid cannot be detected in daughter cells after a sufficient number ofcell divisions. Both transiently transformed and stably transformedtransgenic plants and plant cells can be useful in the methods describedherein.

Transgenic plant cells used in methods described herein can constitutepart or all of a whole plant. Such plants can be grown in a mannersuitable for the species under consideration, either in a growthchamber, a greenhouse, or in a field. Transgenic plants can be bred asdesired for a particular purpose, e.g., to introduce a heterologousnucleic acid, for example a recombinant nucleic acid construct intoother lines, to transfer a heterologous nucleic acid to other species,or for further selection of other desirable traits. Alternatively,transgenic plants can be propagated vegetatively for those speciesamenable to such techniques. As used herein, a transgenic plant alsorefers to progeny of an initial transgenic plant provided the progenyinherits the transgene. Seeds produced by a transgenic plant can begrown and then selfed (or outcrossed and selfed) to obtain seedshomozygous for the nucleic acid construct.

Transgenic plants can be grown in suspension culture, or tissue or organculture. For the purposes of this invention, solid and/or liquid tissueculture techniques can be used. When using solid medium, transgenicplant cells can be placed directly onto the medium or can be placed ontoa filter that is then placed in contact with the medium. When usingliquid medium, transgenic plant cells can be placed onto a flotationdevice, e.g., a porous membrane that contacts the liquid medium.

When transiently transformed plant cells are used, a reporter sequenceencoding a reporter polypeptide having a reporter activity can beincluded in the transformation procedure and an assay for reporteractivity or expression can be performed at a suitable time aftertransformation. A suitable time for conducting the assay typically isabout 1-21 days after transformation, e.g., about 1-14 days, about 1-7days, or about 1-3 days. The use of transient assays is particularlyconvenient for rapid analysis in different species, or to confirmexpression of a heterologous polypeptide whose expression has notpreviously been confirmed in particular recipient cells.

Techniques for introducing nucleic acids into monocotyledonous anddicotyledonous plants are known in the art, and include, withoutlimitation, Agrobacterium-mediated transformation, viral vector-mediatedtransformation, electroporation and particle gun transformation; seeU.S. Pat. Nos. 5,538,880; 5,204,253; 6,329,571; and 6,013,863. If a cellor cultured tissue is used as the recipient tissue for transformation,plants can be regenerated from transformed cultures if desired, bytechniques known to those skilled in the art.

A population of transgenic plants can be screened and/or selected forthose members of the population that have a trait or phenotype conferredby expression of the transgene. For example, a population of progeny ofa single transformation event can be screened for those plants having adesired level of expression of a polypeptide or nucleic acid describedherein. Physical and biochemical methods can be used to identifyexpression levels. These include Southern analysis or PCR amplificationfor detection of a polynucleotide; northern blots, S1 RNase protection,primer-extension, or RT-PCR amplification for detecting RNA transcripts;enzymatic assays for detecting enzyme or ribozyme activity ofpolypeptides and polynucleotides; and protein gel electrophoresis,western blots, immunoprecipitation, and enzyme-linked immunoassays todetect polypeptides. Other techniques such as in situ hybridization,enzyme staining, and immunostaining also can be used to detect thepresence or expression of polypeptides and/or nucleic acids. Methods forperforming all of the referenced techniques are known.

As an alternative, a population of plants with independenttransformation events can be screened for those plants having a desiredtrait, such as production of vanillin glucoside and lack of vanillicalcohol production. Selection and/or screening can be carried out overone or more generations, and/or in more than one geographic location. Insome cases, transgenic plants can be grown and selected under conditionswhich induce a desired phenotype or are otherwise necessary to produce adesired phenotype in a transgenic plant. In addition, selection and/orscreening can be applied during a particular developmental stage inwhich the phenotype is expected to be exhibited by the plant.

Depending on the particular organism used in this invention, therecombinant host cell can naturally or recombinantly express genesencoding an AROM (Arom Multifunctional Enzyme), OMT(O-methyltransferase), COMT (Catechol-O-Methyl Transferase), 3DSD(3-dehydroshikimate dehydratase), ACAR (aromatic carboxylic acidreductase), UGT (uridine 5′-diphosphoglucosyl transferase), or PPTase(phosphopantetheine transferase) (FIG. 1).

Recombinant expression means that the genome of a host cell has beenaugmented through the introduction of one or more recombinant genes,which include regulatory sequences that facilitate the transcription andtranslation of a protein of interest. While embodiments include stableintroduction of recombinant genes into the host genome, autonomous orreplicative plasmids or vectors can also be used within the scope ofthis invention. Moreover, the present invention can be practiced using alow copy number, e.g., a single copy, or high copy number (asexemplified herein) plasmid or vector.

Generally, the introduced recombinant gene is not originally resident inthe host that is the recipient of the recombinant gene, but it is withinthe scope of the invention to isolate a DNA segment from a given host,and to subsequently introduce one or more additional copies of that DNAinto the same host, e.g., to enhance production of the product of a geneor alter the expression pattern of a gene. In some instances, theintroduced DNA will modify or even replace an endogenous gene or DNAsequence by, e.g., homologous recombination or site-directedmutagenesis. Suitable recombinant hosts include microorganisms, plantcells, and plants.

The term “recombinant gene” refers to a gene or DNA sequence that isintroduced into a recipient host, regardless of whether the same or asimilar gene or DNA sequence may already be present in such a host.“Introduced,” or “augmented” in this context, is known in the art tomean introduced or augmented by the hand of man. Thus, a recombinantgene may be a DNA sequence from another species, or may be a DNAsequence that originated from or is present in the same species, but hasbeen incorporated into a host by recombinant methods to form arecombinant host. It will be appreciated that a recombinant gene that isintroduced into a host can be identical to a DNA sequence that isnormally present in the host being transformed, and is introduced toprovide one or more additional copies of the DNA to thereby permitoverexpression or modified expression of the gene product of that DNA.

A recombinant gene encoding a polypeptide described herein includes thecoding sequence for that polypeptide, operably linked, in senseorientation, to one or more regulatory regions suitable for expressingthe polypeptide. Because many microorganisms are capable of expressingmultiple gene products from a polycistronic mRNA, multiple polypeptidescan be expressed under the control of a single regulatory region forthose microorganisms, if desired. A coding sequence and a regulatoryregion are considered to be operably linked when the regulatory regionand coding sequence are positioned so that the regulatory region iseffective for regulating transcription or translation of the sequence.Typically, the translation initiation site of the translational readingframe of the coding sequence is positioned between one and about fiftynucleotides downstream of the regulatory region for a monocistronicgene.

In many cases, the coding sequence for a polypeptide described herein isidentified in a species other than the recombinant host, i.e., is aheterologous nucleic acid. The term “heterologous nucleic acid” as usedherein, refers to a nucleic acid introduced into a recombinant host,wherein said nucleic acid is not naturally present in said host. Thus,if the recombinant host is a microorganism, the coding sequence can befrom other prokaryotic or eukaryotic microorganisms, from plants or fromanimals. n some case, however, the coding sequence is a sequence that isnative to the host and is being reintroduced into that organism. Anative sequence can often be distinguished from the naturally occurringsequence by the presence of non-natural sequences linked to theexogenous nucleic acid, e.g., non-native regulatory sequences flanking anative sequence in a recombinant nucleic acid construct. In addition,stably transformed exogenous nucleic acids typically are integrated atpositions other than the position where the native sequence is found.

“Regulatory region” refers to a nucleic acid having nucleotide sequencesthat influence transcription or translation initiation and rate, andstability and/or mobility of a transcription or translation product.Regulatory regions include, without limitation, promoter sequences,enhancer sequences, response elements, protein recognition sites,inducible elements, protein binding sequences, 5′ and 3′ untranslatedregions (UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, introns, and combinations thereof. Aregulatory region typically includes at least a core (basal) promoter. Aregulatory region also may include at least one control element, such asan enhancer sequence, an upstream element or an upstream activationregion (UAR). A regulatory region is operably linked to a codingsequence by positioning the regulatory region and the coding sequence sothat the regulatory region is effective for regulating transcription ortranslation of the sequence. For example, to operably link a codingsequence and a promoter sequence, the translation initiation site of thetranslational reading frame of the coding sequence is typicallypositioned between one and about fifty nucleotides downstream of thepromoter. A regulatory region can, however, be positioned as much asabout 5,000 nucleotides upstream of the translation initiation site, orabout 2,000 nucleotides upstream of the transcription start site.

The choice of regulatory regions to be included depends upon severalfactors, including, but not limited to, efficiency, selectability,inducibility, desired expression level, and preferential expressionduring certain culture stages. It is a routine matter for one of skillin the art to modulate the expression of a coding sequence byappropriately selecting and positioning regulatory regions relative tothe coding sequence. It will be understood that more than one regulatoryregion may be present, e.g., introns, enhancers, upstream activationregions, transcription terminators, and inducible elements.

One or more genes, for example one or more heterologous nucleic acids,can be combined in a recombinant nucleic acid construct in “modules”useful for a discrete aspect of vanillin and/or vanillin glucosideproduction. Combining a plurality of genes or heterologous nucleic acidsin a module, facilitates the use of the module in a variety of species.For example, a vanillin gene cluster can be combined such that eachcoding sequence is operably linked to a separate regulatory region, toform a vanillin module for production in eukaryotic organisms.Alternatively, the module can express a polycistronic message forproduction of vanillin and/or vanillin glucoside in prokaryotic hostssuch as species of Rodobacter, E. coli, Bacillus or Lactobacillus. Inaddition to genes useful for vanillin or vanillin glucoside production,a recombinant construct typically also contains an origin ofreplication, and one or more selectable markers for maintenance of theconstruct in appropriate species.

It will be appreciated that because of the degeneracy of the geneticcode, a number of nucleic acids can encode a particular polypeptide;i.e., for many amino acids, there is more than one nucleotide tripletthat serves as the codon for the amino acid. Thus, codons in the codingsequence for a given polypeptide can be modified such that optimalexpression in a particular host is obtained, using appropriate codonbias tables for that host (e.g., microorganism). As isolated nucleicacids, these modified sequences can exist as purified molecules and canbe incorporated into a vector or a virus for use in constructing modulesfor recombinant nucleic acid constructs.

As indicated, recombinant hosts can express one or more enzymes involvedin the biosynthesis of the vanillin or vanillin glucoside, as well asadditional genes or biosynthetic modules that improve efficiency withwhich energy and carbon sources are converted to vanillin and itsglucoside, and/or to enhance productivity from the cell culture orplant. In certain embodiments, the recombinant host endogenously orerecombinantly expresses genes encoding AROM, COMT, 3DSD, ACAR, UGT,and/or PPTase.

AROM is a penta-functional enzyme complex encoded in yeast by the ARO1gene. The gene is 4764 bp long and encodes a corresponding polypeptide1588 amino acids in length. AROM performs five consecutive enzymaticconversions, i.e., converting DAHP (3-deoxy-D-arabino-heptulosonicacid-7-phosphate) into 3-DHQ (3-dehydroquinate), which is converted to3-DHS (3-dehydroshikimic acid), which is converted to shikimate, whichis converted to shikimate-3-P (shikimate 3-phosphate), which isconverted into EPSP (5-enolpyruvylskimate 3-phosphate), all en route tocellular biosynthesis of the aromatic amino acids tyrosine, tryptophanand phenylalanine. According to some embodiments of this invention, theAROM enzyme possesses at least four of the five enzymatic activities ofthe S. cerevisiae AROM polypeptide, i.e., 3-dehydroquinate dehydrataseactivity, 3-dehydroquinate synthase activity, 3-phosphoshikimate1-carboxyvinyltransferase activity, shikimate 3-dehydrogenase (NADP+)activity, and shikimate kinase activity.

Non-limiting examples of AROM polypeptides include the Saccharomycescerevisiae polypeptide available under GENBANK Accession No. X06077; theSchizosaccharomyces pombe polypeptide available under GENBANK AccessionNo. NP_594681.1; Schizosaccharomyces japonicas polypeptide availableunder GENBANK Accession No. XP_002171624; Neurospora crassa polypeptideavailable under GENBANK Accession No. XP_956000; and the Yarrowialipolytica polypeptide available under GENBANK Accession No. XP_505337.

According to one embodiment of this invention, the AROM polypeptide is amutant AROM polypeptide with decreased shikimate dehydrogenase activity.When expressed in a recombinant host, the mutant AROM polypeptideredirects metabolic flux from aromatic amino acid production to vanillinprecursor production, i.e., 3-DHS. See WO 2013/022881. In certainembodiments, the mutant AROM polypeptide described herein can have oneor more modifications in domain 5 (e.g., a substitution of one or moreamino acids, a deletion of one or more amino acids, insertions of one ormore amino acids, or combinations of substitutions, deletions, andinsertions).

In some embodiments, a modified AROM polypeptide is fused to apolypeptide catalyzing the first committed step of vanillinbiosynthesis, 3DSD. A polypeptide having 3DSD activity and that issuitable for use in a fusion polypeptide includes the 3DSD polypeptidefrom Podospora pauciseta, Ustilago maydis, Rhodoicoccus jostii,Acinetobacter sp., Aspergillus niger or Neurospora crassa. See, GENBANKAccession Nos. CAD60599), XP_001905369.1, XP_761560.1, ABG93191.1,AAC37159.1, and XM_001392464.

Alternatively, or in addition to, the recombinant host can express aCOMT polypeptide. Non-limiting examples of COMT polypeptides of use inthis invention include COMT polypeptides in the family classified underEC number 2.1.1.6, such as the Homo sapiens (Hs) polypeptide availableunder GENBANK Accession No. NM 000754; an Arabidopsis thalianapolypeptide available under GENBANK Accession No. AY062837; or aFragaria x ananassa (strawberry) polypeptide available under GENBANKAccession No. AF220491. Human COMT polypeptide exists as severalvariants and the COMT polypeptide may be any of these variants. Othersuitable mammalian COMT polypeptides of use in this invention include,but are not limited to, those isolated from Pan troglodytes (GENBANKAccession No. XP_514984), Macaca mulatta (GENBANK Accession No.AFJ70145), Equus caballus (GENBANK Accession No. NP_001075303), Canislupus familiaris (GENBANK Accession No. AAR20324), Cricetulus griseus(GENBANK Accession No. EGV97595), Sus scrofa (GENBANK Accession No.NP_001182259), and Bos Laurus (GENBANK Accession No. NP 001095787).Other exemplary COMT polypeptides from plant and microorganism sourcesinclude, but are not limited to, those isolated from Rosa chinensis(GENBANK Accession No. CAD29457), Prunus dulcis (GENBANK Accession No.CAA58218), Gossypium hirsutum (GENBANK Accession No. ACT32028), Jatrophacurcas (GENBANK Accession No. ACT87981), Eucalyptus camaldulensis(ADB82906), Candida orthopsilosis (GENBANK Accession No. CCG25047),Pichia stipitis (GENBANK Accession No. ABN67921), and Spathasporapassalidarum (GENBANK Accession No. EGW29958). In certain embodiments,the COMT polypeptide of the invention is obtained from Phytophtherainfestans (GENBANK Accession No. XP 002899214), Catharanthus roseus(GENBANK Accession No. EGS21863), Yarrowia lipolytica (GENBANK AccessionNo. XP 500451), Ciona intestinalis (GENBANK Accession No. XP 002121420or XP 002131313), Capsasproa owczarzaki (GENBANK Accession No.EFW46044), Chaetomium therophilum (GENBANK Accession No. EGS21863),Clavispora lusitaniae (GENBANK Accession No. XP 002899214),Paracoccidioides sp. ‘lutzii’ Pb01 (GENBANK Accession No. XP_002793380),Vanilla planifolia (see SEQ ID NO:56 of PCT/US2012/049842), CoffeaArabica (GENBANK Accession No. AAN03726), Rattus norvegicus (GENBANKAccession No. NP_036663), Mus musculus (GENBANK Accession No. NP031770), Crenarchaeote (GENBANK Accession No. ABZ07345), Mycobacteriumvanbaleeni (GENBANK Accession No. ABM14078), or Schizosaccharomycespombe (GENBANK Accession No. NP_001018770.

In some embodiments, a mutant COMT polypeptide is used to improvebiosynthesis of vanillin beta-D-glucoside. For example, mutant COMTpolypeptides can have one or more of the following properties: increasedturnover; preferential methylation at the meta (3′) position, ratherthan at the para (4′) position such that production of vanillin isfavored over isovanillin; or better specificity for the vanillin pathwaysubstrates, protocatechuic acid and protocatechuic aldehyde. See WO2013/022881. A mutant COMT polypeptide can have one or more mutations(e.g., a substitution of one or more amino acids, a deletion of one ormore amino acids, insertions of one or more amino acids, or combinationsof substitutions, deletions, and insertions) in, for example, thesubstrate binding site. For example, a mutant COMT polypeptide can haveone or more amino acid substitutions in the substrate binding site ofhuman COMT.

In one embodiment, a mutant COMT polypeptide is provided, which iscapable of catalyzing methylation of an —OH group of protocatechuicacid, wherein said methylation results in generation of at least 4 timesmore vanillic acid compared to iso-vanillic acid. In another embodiment,the mutant COMT polypeptide is capable of catalyzing methylation of an—OH group of protocatechuic aldehyde, wherein said methylation resultsin generation of at least 4 times more vanillin compared toiso-vanillin.

In some embodiments, the host harbors a nucleic acid encoding mutantAROM polypeptide and optionally a wild-type COMT polypeptide. In anotherembodiment, the host of this invention harbors a nucleic acid encodingmutant COMT polypeptide and optionally a wild-type AROM polypeptide. Inyet another embodiment, the host of this invention harbors a nucleicacid encoding mutant AROM polypeptide and optionally a mutant COMTpolypeptide.

Suitable 3DSD polypeptides are known. A 3DSD polypeptide according tothe present invention may be any enzyme with 3-dehydroshikimatedehydratase activity. Preferably, the 3DSD polypeptide is an enzymecapable of catalyzing conversion of 3-dehydro-shikimate toprotocatechuate and H₂O. A 3DSD polypeptide according to the presentinvention is preferably an enzyme classified under EC 4.2.1.118. Forexample, a suitable polypeptide having 3DSD activity includes the 3DSDpolypeptide made by Podospora pauciseta, Ustilago maydis, Rhodoicoccusjostii, Acinetobacter sp., Aspergillus niger or Neurospora crassa. See,GENBANK Accession Nos. CAD60599, XP_001905369.1, XP_761560.1,ABG93191.1, AAC37159.1, and XM_001392464. Thus, the recombinant host mayinclude a heterologous nucleic acid encoding the 3DSD polypeptide ofPodospora anserina, Ustilago maydis, Rhodoicoccus jostii, Acinetobactersp., Aspergillus niger or Neurospora crassa or a functional homologue ofany of the aforementioned sharing at least 80%, such as at least 85%,for example at least 90%, such as at least 95%, for example at least 98%sequence identity therewith.

Suitable ACAR polypeptides are also known in the art. An ACARpolypeptide according to the present invention may be any enzyme havingaromatic carboxylic acid reductase activity. Preferably, the ACARpolypeptide is an enzyme capable of catalyzing conversion protocatechuicacid to protocatechuic aldehyde and/or conversion of vanillic acid tovanillin. An ACAR polypeptide according to the present invention ispreferably an enzyme classified under EC 1.2.1.30. For example asuitable ACAR polypeptide is made by Nocardia sp. See, e.g., GENBANKAccession No. AY495697. Thus, the recombinant host may include aheterologous nucleic acid encoding the ACAR polypeptide of Nocardia sp.or a functional homologue thereof sharing at least 80%, such as at least85%, for example at least 90%, such as at least 95%, for example atleast 98% sequence identity therewith.

Suitable PPTase polypeptides are known. A PPTase polypeptide accordingto the present invention may be any enzyme capable of catalyzingphosphopantetheinylation. Preferably, the PPTase polypeptide is anenzyme capable of catalyzing phosphopantetheinylation of ACAR. Forexample, a suitable PPTase polypeptide is made by E. coli,Corynebacterium glutamicum, or Nocardia farcinica. See GENBANK AccessionNos. NP_601186, BAA35224, and YP_120266. Thus, the recombinant host mayinclude a heterologous nucleic acid encoding the PPTase polypeptide ofE. coli, C. glutamicum, or N. farcinica or a functional homologue of anyof the aforementioned sharing at least 80%, such as at least 85%, forexample at least 90%, such as at least 95%, for example at least 98%sequence identity therewith.

Glucosylation of vanillin is particularly useful. Vanillin-β-D-glucosideis the storage form of vanillin found in the vanilla pod. It isnon-toxic to most organisms, including yeast, and has a highersolubility in water, as compared to vanillin. In addition, the formationof vanillin-β-D-glucoside most likely directs the biosynthesis towardvanillin production. UGT72E2 (Hansen, et al. (2009) Appl. Environ.Microbiol. 75:2765-27740) exhibited high substrate specificity towardvanillin. In concordance with this observation, its expression in thevanillin producing S. cerevisiae strain resulted in almost all vanillinbeing converted into vanillin-β-D-glucoside. The ability to turnvanillin into vanillin-β-D-glucoside in vivo is important, becausemicrobial production of non-glucosylated vanillin beyond the 0.5-1g/liter scale would be hampered by the toxicity of free vanillin.Glucosylation serves to circumvent the inhibitory effect.

Accordingly, the recombinant host of this invention can also express aUGT polypeptide. A UGT polypeptide may be any UDP-Glucose:Aglycon-Glucosyltransferase. Preferably the UGT polypeptides cancatalyze the glucosylation of vanillin (i.e., to produce vanillinbeta-D-glucoside). Thus, the UGT polypeptide may be a Family 1glycosyltransferease. Preferred UGT polypeptides according to theinvention are classified under EC 2.4.1. Suitable UGT polypeptidesinclude the UGT71C2, UGT72B1, UGT72E2, UGT84A2, UGT89B1, UGT85B1, andarbutin synthase polypeptides. See, e.g., GENBANK Accession Nos.AC0005496, NM_116337, and NM_126067. The A. thaliana UGT72E2 isparticularly useful (see, e.g., Hansen, et al. (2009) supra). Thus, therecombinant host may include a heterologous nucleic acid encoding theUGT71C2, UGT72B1, UGT72E2, UGT84A2, UGT89B1, UGT85B1, or arbutinsynthase or a functional homologue of any of the aforementioned sharingat least 80%, such as at least 85%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith. Otheruseful UGTs are described in WO 01/40491.

As a further embodiment of this invention, a VAO enzyme (EC 1.1.3.38)can also be expressed by host cells to oxidize any residual vanillylalcohol into vanillin. VAO enzymes are known in the art and include, butare not limited to enzymes from filamentous fungi such as Fusariummonilifomis (GENBANK Accession No. AFJ11909) and Penicilliumsimplicissium (GENBANK Accession No. P56216; Benen, et al. (1998) J.Biol. Chem. 273:7865-72) and bacteria such as Modestobacter marinus(GENBANK Accession No. YP_006366868), Rhodococcus jostii (GENBANKAccession No. YP_703243.1) and R. opacus (GENBANK Accession No.EHI39392).

In some cases, it is desirable to inhibit one or more functions of anendogenous polypeptide in order to divert metabolic intermediates towardvanillin or vanillin glucoside biosynthesis. For example, pyruvatedecarboxylase (PDC1) and/or glutamate dehydrogenase activity can bereduced. In such cases, a nucleic acid that inhibits expression of thepolypeptide or gene product may be included in a recombinant constructthat is transformed into the strain. Alternatively, mutagenesis can beused to generate mutants in genes for which it is desired to inhibitfunction.

To demonstrate expression and activity of one or more of theabove-referenced enzymes expressed by the recombinant host, levels ofproducts, substrates and intermediates, e.g., dehydroshikimic acid,protocatechuic acid, protocatechuic aldehyde, vanillin, and vanillinbeta-D-glucoside produced by the recombinant host can be determined byextracting samples from culture media for analysis according topublished methods.

Recombinant hosts described herein can be used in methods to producevanillin or vanillin glucoside. For example, if the recombinant host isa microorganism, the method can include growing the recombinantmicroorganism in a culture medium under conditions in which vanillinand/or vanillin glucoside biosynthesis genes are expressed. Therecombinant microorganism may be grown in a batch, fed batch orcontinuous process or combinations thereof. Typically, the recombinantmicroorganism is grown in a fermentor at a defined temperature(s) in thepresence of a suitable nutrient source, e.g., a carbon source, for adesired period of time to produce a desired amount of vanillin and/orvanillin glucoside.

Therefore, this invention also provides a method for producing vanillinand/or vanillin beta-D-glucoside by providing a recombinant host thatproduces vanillin and/or vanillin beta-D-glucoside and has reducedproduction or activity of at least one (or two, three, four, five, six,seven, eight, nine or ten) alcohol dehydrogenase, at least one aldehydereductase, or at least one alcohol dehydrogenase and at least onealdehyde reductase; cultivating said recombinant host, e.g., in thepresence of a suitable carbon source, for a time sufficient for saidrecombinant host to produce vanillin and/or vanillin glucoside; andisolating vanillin and/or vanillin glucoside from said recombinant hostor from the cultivation supernatant. In one embodiment, the recombinanthost produces a reduced amount of vanillic alcohol in comparison to ahost that expresses the one or more functional alcohol dehydrogenases orone or more aldehyde reductases.

In certain embodiments, it is preferred that the recombinant hostexpresses at least one 3DSD and at least one ACAR, which preferably maybe one of the 3DSD's and ACAR's described herein. In embodiments wherethe recombinant host expresses an ACAR capable of catalyzing conversionof vanillic acid to vanillin, then the method may also includedetermining the level of generated vanillin and iso-vanillin. Therecombinant host may also express at least one UGT capable of catalyzingglucosylation of vanillin and isovanillin, in which case the levels ofvanillin-glucoside and iso-vanillin-glucoside may be determined insteadof the levels of vanillin and iso-vanillin, respectively. Alternatively,this may be determined by generating a recombinant host harboring aheterologous nucleic acid encoding the mutant COMT polypeptide to betested, and feeding protocatechuic acid to said recombinant host,followed by determining the level of generated iso-vanillic acid andvanillic acid.

Similarly, an in vitro assay or a recombinant host cell can be used todetermine whether a mutant COMT polypeptide is capable of catalyzingmethylation of an —OH group of protocatechuic aldehyde, wherein saidmethylation results in generation of at least X times more vanillincompared to iso-vanillin. However, in this assay, protecatechuicaldehyde is used as starting material and the level of vanillin andiso-vanillin is determined.

Likewise, an in vitro assay or a recombinant host cell can be used todetermine whether a given mutant COMT polypeptide is capable ofcatalyzing methylation of an —OH group of protocatechuic alcohol,wherein said methylation results in generation of at least X times morevanillyl alcohol compared to iso-vanillyl alcohol. However, in thisassay, protecatechuic alcohol is used as starting material and the levelof vanillyl alcohol and iso-vanillyl alcohol is determined.

The level of isovanillin and vanillin may be determined by any suitablemethod useful for detecting these compounds, wherein said method candistinguish between isovanillin and vanillin. Such methods include forexample HPLC. Similarly, the level of iso-vanillic acid, vanillic acid,iso-vanillyl alcohol and vanillyl alcohol may be determined using anysuitable method useful for detecting these compounds, wherein saidmethod can distinguish between isovanillin and vanillin. Such methodsinclude for example HPLC.

Carbon sources of use in the method of this invention include anymolecule that can be metabolized by the recombinant host cell tofacilitate growth and/or production of the vanillin and/or vanillinglucoside. Examples of suitable carbon sources include, but are notlimited to, sucrose (e.g., as found in molasses), fructose, xylose,ethanol, glycerol, glucose, cellulose, starch, cellobiose or otherglucose containing polymer. In embodiments employing yeast as a host,for example, carbons sources such as sucrose, fructose, xylose, ethanol,glycerol, and glucose are suitable. The carbon source can be provided tothe host organism throughout the cultivation period or alternatively,the organism can be grown for a period of time in the presence ofanother energy source, e.g., protein, and then provided with a source ofcarbon only during the fed-batch phase.

After the recombinant host has been grown in culture for the desiredperiod of time, vanillin and/or vanillin beta-D-glucoside can then berecovered from the culture using various techniques known in the art,e.g., isolation and purification by extraction, vacuum distillation andmulti-stage re-crystallization from aqueous solutions andultrafiltration (Böddeker, et al. (1997) J. Membrane Sci. 137:155-158;Borges da Silva, et al. (2009) Chem. Eng. Des. 87:1276-1292). Two-phaseextraction processes, employing either sulphydryl compounds, such asdithiothreitol, dithioerythritol, glutathione, or L-cysteine (U.S. Pat.No. 5,128,253), or alkaline KOH solutions (WO 94/13614), have been usedin the recovery of vanillin as well as for its separation from otheraromatic substances. Vanillin adsorption and pervaporation frombioconverted media using polyether-polyamide copolymer membranes hasalso been described (Boddeker, et al. (1997) supra; Zucchi, et al.(1998) J. Microbiol. Biotechnol. 8:719-722). Macroporous adsorptionresins with crosslinked-polystyrene framework have also been used torecover dissolved vanillin from aqueous solutions (Zhang, et al. (2008)Eur. Food Res. Technol. 226:377-383). Ultrafiltration and membranecontactor (MC) techniques have also been evaluated to recover vanillin(Zabkova, et al. (2007) J. Membr. Sci. 301:221-237; Scuibba, et al.(2009) Desalination 241:357-364). Alternatively, conventional techniquessuch as percolation or supercritical carbon dioxide extraction andreverse osmosis for concentration could be used. If the recombinant hostis a plant or plant cells, vanillin or vanillin glucoside can beextracted from the plant tissue using various techniques known in theart.

In some embodiments, vanillin or vanillin beta-D-glucoside can beproduced using whole cells that are fed raw materials that containprecursor molecules. The raw materials may be fed during cell growth orafter cell growth. The whole cells may be in suspension or immobilized.The whole cells may be in fermentation broth or in a reaction buffer. Insome embodiments a permeabilizing agent may be required for efficienttransfer of substrate into the cells.

In some embodiments, the vanillin or vanillin beta-D-glucoside isisolated and purified to homogeneity (e.g., at least 90%, 92%, 94%, 96%,or 98% pure). In other embodiments, the vanillin or vanillinbeta-D-glucoside is isolated as an extract from a recombinant host. Inthis respect, vanillin or vanillin beta-D-glucoside may be isolated, butnot necessarily purified to homogeneity. Desirably, the amount ofvanillin or vanillin beta-D-glucoside produced can be from about 1 mg/lto about 20,000 mg/L or higher. For example about 1 to about 100 mg/L,about 30 to about 100 mg/L, about 50 to about 200 mg/L, about 100 toabout 500 mg/L, about 100 to about 1,000 mg/L, about 250 to about 5,000mg/L, about 1,000 to about 15,000 mg/L, or about 2,000 to about 10,000mg/L of vanillin or vanillin beta-D-glucoside can be produced. Ingeneral, longer culture times will lead to greater amounts of product.Thus, the recombinant microorganism can be cultured for from 1 day to 7days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4days, or about 5 days.

It will be appreciated that the various genes and modules discussedherein can be present in two or more recombinant microorganisms ratherthan a single microorganism. When a plurality of recombinantmicroorganisms is used, they can be grown in a mixed culture to producevanillin and/or vanillin glucoside.

Extracts of isolated, and optionally purified, vanillin or vanillinbeta-D-glucoside find use in flavoring consumables such as foodproducts, dietary supplements, nutraceuticals, pharmaceuticalcompositions, dental hygienic compositions, and cosmetic products.

The phrase “food product,” as used herein, includes, but is not limitedto, fruits, vegetables, juices, meat products such as ham, bacon andsausage; egg products, fruit concentrates, gelatins and gelatin-likeproducts such as jams, jellies, preserves, and the like; milk productssuch as ice cream, sour cream and sherbet; icings, syrups includingmolasses; corn, wheat, rye, soybean, oat, rice and barley products, nutmeats and nut products, cakes, cookies, confectionaries such as candies,gums, fruit flavored drops, and chocolates, chewing gum, mints, creams,icing, ice cream, pies and breads, beverages such as coffee, tea,carbonated soft drinks, such as COKE and PEPSI, non-carbonated softdrinks, juices and other fruit drinks, sports drinks such as GATORADE,coffee, teas, iced teas, cola, alcoholic beverages, such as beers, winesand liquors, and KOOL-AID.

Food products also include condiments such as herbs, spices andseasonings, flavor enhancers. A food product also includes preparedpackaged products, such as dietetic sweeteners, liquid sweeteners,granulated flavor mixes which upon reconstitution with water providenon-carbonated drinks, instant pudding mixes, instant coffee and tea,coffee whiteners, malted milk mixes, pet foods, livestock feed, tobacco,and materials for baking applications, such as powdered baking mixes forthe preparation of breads, cookies, cakes, pancakes, donuts and thelike. Food products also include diet or low-calorie food and beveragescontaining little or no sucrose. Other examples of food productsenvisioned in accordance with the present invention are described belowand throughout the specification.

In another embodiment, the food products are fruits, vegetables, juices,meat products such as ham, bacon and sausage; egg products, fruitconcentrates, gelatins and gelatin-like products such as jams, jellies,preserves, and the like; milk products such as ice cream, sour cream andsherbet; icings, syrups including molasses; corn, wheat, rye, soybean,oat, rice and barley products, nut meats and nut products, cakes,cookies, confectionaries such as candies, gums, fruit flavored drops,and chocolates, creams, icing, ice cream, pies and breads.

In another embodiment, the consumable is a pharmaceutical composition.Preferred compositions are pharmaceutical compositions containingvanillin and/or vanillin beta-D-glucoside and one or morepharmaceutically acceptable excipients. These pharmaceuticalcompositions can be used to formulate pharmaceutical drugs containingone or more active agents that exert a biological effect. As such, thepharmaceutical composition preferably further include one or more activeagents that exert a biological effect. Such active agents includepharmaceutical and biological agents that have an activity. Such activeagents are well known in the art. See, e.g., The Physician's DeskReference. Such compositions can be prepared according to proceduresknown in the art, for example, as described in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., USA. In oneembodiment, such an active agent includes bronchodilators, anorexiants,antihistamines, nutritional supplements, laxatives, analgesics,anesthetics, antacids, H2-receptor antagonists, anticholinergics,antidiarrheals, demulcents, antitussives, antinauseants, antimicrobials,antibacterials, antifungals, antivirals, expectorants, anti-inflammatoryagents, antipyretics, and mixtures thereof. In one embodiment, theactive agent is an antipyretics or analgesics, e.g., ibuprofen,acetaminophen, or aspirin; laxatives, e.g., phenolphthalein dioctylsodium sulfosuccinate; appetite depressants, e.g., amphetamines,phenylpropanolamine, phenylpropanolamine hydrochloride, or caffeine;antacidics, e.g., calcium carbonate; antiasthmatics, e.g., theophylline;antidiuretics, e.g., diphenoxylate hydrochloride; agents active againstflatulence, e.g., simethecon; migraine agents, e.g., ergotaminetartrate;psychopharmacological agents, e.g., haloperidol; spasmolytics orsedatives, e.g., phenobarbitol; antihyperkinetics, e.g., methyldopa ormethylphenidate; tranquilizers, e.g., benzodiazepines,hydroxinmeprobramates or phenothiazines; antihistaminics, e.g.,astemizol, chloropheniramine maleate, pyridamine maleate, doxlaminesuccinate, bromopheniramine maleate, phenyltoloxamine citrate,chlorocyclizine hydrochloride, pheniramine maleate, and phenindaminetartrate; decongestants, e.g., phenylpropanolamine hydrochloride,phenylephrine hydrochloride, pseudoephedrine hydrochloride,pseudoephedrine sulfate, phenylpropanolamine bitartrate, and ephedrine;beta-receptor blockers, e.g., propanolol; agents for alcohol withdrawal,e.g., disulfiram; antitussives, e.g., benzocaine, dextromethorphan,dextromethorphan hydrobromide, noscapine, carbetapentane citrate, andchlophedianol hydrochloride; fluorine supplements, e.g., sodiumfluoride; local antibiotics, e.g., tetracycline or cleocine;corticosteroid supplements, e.g., prednisone or prednisolone; agentsagainst goiter formation, e.g., colchicine or allopurinol;antiepileptics, e.g., phenytoine sodium; agents against dehydration,e.g., electrolyte supplements; antiseptics, e.g., cetylpyridiniumchloride; NSAIDs, e.g., acetaminophen, ibuprofen, naproxen, or saltsthereof; gastrointestinal active agents, e.g., loperamide andfamotidine; various alkaloids, e.g., codeine phosphate, codeine sulfate,or morphine; supplements for trace elements, e.g., sodium chloride, zincchloride, calcium carbonate, magnesium oxide, and other alkali metalsalts and alkali earth metal salts; vitamins; ion-exchange resins, e.g.,cholestyramine; cholesterol-depressant and lipid-lowering substances;antiarrhythmics, e.g., N-acetylprocainamide; and expectorants, e.g.,guaifenesin.

Active substances which have a particularly unpleasant taste includeantibacterial agents such as ciprofloxacin, ofloxacin, and pefloxacin;antiepileptics such as zonisamide; macrolide antibiotics such aserythromycin; beta-lactam antibiotics such as penicillins andcephalosporins; psychotropic active substances such as chlorpromazine;active substances such as sulpyrine; and agents active against ulcers,such as cimetidine.

The pharmaceutical compositions of this invention are administered to asubject in any form suitable to achieve their intended purpose.Preferably, however, the composition is one which can be administeredbuccally or orally. Alternatively, the pharmaceutical composition can bean oral or nasal spray. The subject is any animal, such as a human,although the invention is not intended to be so limited. Other suitableanimals include canines, felines, dogs, cats, livestock, horses, cattle,sheep, and the like. A veterinary composition, as used herein, refers toa pharmaceutical composition that suitable for non-human animals. Suchveterinary compositions are known in the art.

In another embodiment, the pharmaceutical composition is a liquid dosageform for oral administration, including pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and elixirs. In addition tothe active compounds, the liquid dosage forms can contain inert diluentscommonly used in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide,oils (in particular, cottonseed, groundnut, corn, germ, olive, castor,and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions, in addition to the active compounds, can contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, and tragacanth, and mixturesthereof.

The pharmaceutical composition of the present invention can be in theform of a chewable tablet. Chewable tablets are known in the art. See,e.g., U.S. Pat. Nos. 4,684,534 and 6,060,078, each of which isincorporated by reference in its entirety. Any kind of medicament can becontained in the chewable tablet, preferably a medicament of bittertaste, natural plant extracts or other organic compounds. Morepreferably, vitamins such as vitamin A, vitamin B, vitamin B1, vitaminB2, vitamin B6, vitamin C, vitamin E and vitamin K; natural plantextracts such as Sohgunjung-tang extracts, Sipchundaebo-tang extractsand Eleutherococcus senticosus extracts; organic compounds such asdimenhydrinate, meclazine, acetaminophen, aspirin, phenylpropanolamine,and cetylpyridinium chloride; or gastrointestinal agents such as driedaluminum hydroxide gel, domperidone, soluble azulene, L-glutamine andhydrotalcite can be contained in the core.

The pharmaceutical composition of the present invention can be an orallydisintegrating composition. Orally disintegrating tablets are known inthe art. See, e.g., U.S. Pat. Nos. 6,368,625 and 6,316,029, each ofwhich is hereby incorporated by reference in its entirety.

The pharmaceutical composition of the present invention can be a soliddosage form, including vanillin or vanillin beta-D-glucoside and a waterand/or saliva activated effervescent granule, such as one having acontrollable rate of effervescence. The effervescent composition canfurther comprise a pharmaceutically active compound. Effervescentpharmaceutical compositions are known in the art. See, e.g., U.S. Pat.No. 6,649,186, which is incorporated by reference in its entirety. Theeffervescent composition can be used in pharmaceutical, veterinary,horticultural, household, food, culinary, pesticidal, agricultural,cosmetic, herbicidal, industrial, cleansing, confectionery and flavoringapplications. Formulations incorporating the effervescent compositioncontaining vanillin or vanillin beta-D-glucoside can further include oneor more additional adjuvants and/or active ingredients which can bechosen from those known in the art, including flavors, diluents, colors,binders, filler, surfactant, disintegrant, stabilizer, compactionvehicles, and non-effervescent disintegrants.

The pharmaceutical composition can be a film-shaped or wafer-shapedpharmaceutical composition. Such a film-shaped or wafer-shapedpharmaceutical composition can be configured, for example, as quicklydisintegrating administration forms, e.g., administration formsdisintegrating within a period of 1 second up to 3 minutes, or as slowlydisintegrating administration forms, e.g., administration formsdisintegrating within a period of 3 to 15 minutes. The indicateddisintegration times can be set to the above-mentioned ranges by using,for example, matrix-forming polymers which have differentdisintegrating, or solubility, characteristics. Thus, by mixing thecorresponding polymer components, the disintegration time can beadjusted. In addition, disintegrants are known which “draw” water intothe matrix and cause the matrix to burst open from within. As aconsequence, certain embodiments of the invention include suchdisintegrants for the purpose of adjusting the disintegration time.

Suitable are polymers for use in the film-shaped or wafer-shapedpharmaceutical composition include cellulose derivatives, polyvinylalcohol (e.g. MOWIOL), polyacrylates, polyvinyl pyrrolidone, celluloseethers, such as ethyl cellulose, as well as polyvinyl alcohol,polyurethane, polymethacrylates, polymethyl methacrylates andderivatives and copolymerizates of the aforementioned polymers.

In certain embodiments, the total thickness of the film-shaped orwafer-shaped pharmaceutical composition according to the invention ispreferably 5 μm up to 10 mm, preferably 30 μm to 2 mm, and withparticular preference 0.1 mm to 1 mm. The pharmaceutical preparationscan be round, oval, elliptic, triangular, quadrangular or polygonalshape, but they can also have any rounded shape.

The pharmaceutical composition of the present invention can be in theform of an aerosol. The aerosol composition can further include apharmaceutically active agent. Aerosol compositions are known in theart. See, e.g., U.S. Pat. No. 5,011,678, which is hereby incorporated byreference in its entirety. As a nonlimiting example, an aerosolcomposition according to the present invention can include a medicallyeffective amount of a pharmaceutically active substance, vanillin orvanillin beta-D-glucoside and a biocompatible propellant, such as a(hydro/fluoro)carbon propellant.

In one embodiment of the present invention, the pharmaceuticalcomposition is a nutritional composition. Examples of nutritionalcompositions having an undesirable taste include, but are notnecessarily limited to, enteral nutrition products for treatment ofnutritional deficit, trauma, surgery, Crohn's disease, renal disease,hypertension, obesity and the like, to promote athletic performance,muscle enhancement or general well being or inborn errors of metabolismsuch as phenylketonuria. In particular, such nutritional formulationscan contain one or more amino acids which have a bitter or metallictaste or aftertaste. Such amino acids include, but are not limited to,an essential amino acids such as an L isomer of leucine, isoleucine,histidine, lysine, methionine, phenylalanine, threonine, tryptophan,tyrosine or valine.

In one embodiment, the consumable of the present invention is a dentalhygienic composition containing vanillin and/or vanillinbeta-D-glucoside. Dental hygienic compositions are known in the art andinclude, but are not necessarily limited to, toothpaste, mouthwash,plaque rinse, dental floss, dental pain relievers (such as ANBESOL), andthe like.

In another embodiment, the consumable of the present invention is acosmetic product containing vanillin and/or vanillin beta-D-glucoside.For example, but not by way of limitation, the cosmetic product can be aface cream, lipstick, lip gloss, and the like. Other suitablecompositions of the invention include lip balm, such as CHAPSTICK orBURT'S BEESWAX Lip Balm, further containing vanillin and/or vanillinbeta-D-glucoside.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1: Yeast Strains with Reduced Alcohol Dehydrogenase/AldehydeReductase Activity

Yeast strains lacking alcohol dehydrogenase and/or aldehyde reductasewere produced in a parent strain of yeast that was capable of producingvanillin/vanillin beta-D-glucoside. The parent strain, EFSC2932, wascreated by integrating exogenous OMT, UGT and 3DSD genes into the genomeof strain EFSC2055 (Genotype: Mata his3D1 leu2D0 met15D0 ura3D0adh6::LEU2 bg11::KanMX4PTPI1::3DSD[AurC]::(HsOMT::MET15[NatMX])::ACAR[HphMX]::UGT7 2E2[HIS3]ECM3::(CorPPTase-ScHAP4). Genes encoding OMT, UGT and 3DSD wereamplified by polymerase chain reaction (PCR) with gene-specific primersusing X7 DNA polymerase. Vectors for chromosomal integration of geneexpression were constructed using the uracil-specific excision reagent(USER) cloning method and a vector system adapted from Mikkelsen, et al.((2012) Metab. Eng. 14:104-111). The genes were fused to Ashbya gossypiiTEF1 promoter and S. cerevisiae PGK1 promoter by USER cloning. Theresulting constructs were integrated into the following chromosomepositions: XII-1 (pTEF1::HsOMT L198Y::pPGK::HsOMTDNA20), XII-2(pTEF1::HsOMT L198Y+pPGK::UGT72E2) and X-2 (pTEF1::HsOMTL198Y+pPGK::Pa3DSD).

Candidate genes encoding alcohol dehydrogenases or aldehyde reductaseswere selected and knocked out in various combinations. Cultures of eachknockout were analyzed using HPLC-UV to quantify vanillinglucoside/isovanillin glucoside and related products. HPLC analysis wascarried out with an AGILENT 1100 series system with binary pump and aPhenomenex Synergi Polar-RP 2.5 u 100 Å 100×2.00 mm column, whichseparates precursors and Isovanillin and vanillin. A flat gradient wasrun with water/acetonitrile+0.1% trifluoroacetic acid. A 8.9 minuteprogram+1.1 minute postrun was carried out as presented in Table 2.

TABLE 2 Time % Acetonitrile Flow ml/min. 0 5 0.5 0.7 5 0.5 5.7 27 0.56.2 100 0.5 6.6 100 0.7 7.8 100 1.0 8.1 100 1.0 8.6 5 0.8 8.9 5 0.6

Vanillin glucoside and isovanillin glucoside were quantified byintegrating the area of the HPLC peaks and comparing the same with astandard curve. The combinations of genes disrupted and the resultingeffect on vanillyl alcohol production are provided in Table 3.

TABLE 3 Strain Gene Disruption Designation 1 2 3 Result EFSC2906 adh6adh7 adh5 No effect EFSC2907 adh6 adh7 gre3 No effect EFSC2908 adh6 adh7ypr127w No effect EFSC2909 adh6 adh7 ycr102c No effect EFSC2911 adh6adh7 ari1 No effect EFSC2912 adh6 adh7 zta1 No effect EFSC2913 adh6 adh7ycr102c No effect EFSC2929 adh6 ari1 ygl039w Some effect EFSC2930 adh6adh7 ylr460c No effect EFSC2931 adh6 adh7 ygl039w No effect EF5C2932adh6 adh7 gre2 Vanillyl alcohol reduced by 77%

This analysis indicated that, when knocked out, the combination of adh6,adh7 and gre2 provided a 77% decrease in vanillyl alcohol and vanillylalcohol glucoside production (see FIG. 2).

To further analyze the effect of aril, the aril and gre2 genes weredeleted in strain EFSC2055 containing adh6 and adh7 gene knockouts.Strain EFSC2055 and the derived single (EFSC2055 gre2) and double mutant(EFSC 2055 gre2 aril) strains were cultivated for three days inDelft-molasses media (Delft+2% glucose+4% Cane molasses) containing 3 mMof vanillin. EFSC2055 and derived strains encompass theUDP-glycosyltransferase UGT72E2 which is capable of glycosylatingvanillin and vanillyl alcohol. Reduction of vanillin to vanillyl alcoholand vanillyl alcohol glycoside was quantified by HPLC-UV. As shown inFIG. 3, the gre2 deletion alone significantly reduced the formation ofvanillyl alcohol glycoside from vanillin (t-test, P<0.01). However, thedouble deletion of both gre2 and aril in strain EFSC2055 completelyabolished formation of vanillyl alcohol glycoside and significantlyreduced formation of vanillyl alcohol (t-test, P<0.01).

Given the observed effect of aril and YGL039W deletions, it iscontemplated that knocking out one or both of these genes in combinationwith adh6, adh7 or gre2 (e.g., adh6/adh7/gre2/YGL039W;adh6/adh7/gre2/aril/YGL039W; adh6/gre2/aril; adh6/gre2/YGL039W;adh6/gre2/aril/YGL039W) will also yield a decrease in vanillyl alcoholand vanillyl alcohol glucoside production.

What is claimed is:
 1. A recombinant host cell having the followingcharacteristics: (a) the recombinant host cell produces vanillin and/orvanillin glycoside; and (b) the recombinant host cell has a genedisruption or replacement of Alcohol Dehydrogenase 6 (ADH6) gene and agene disruption or replacement of: (i) Aldehyde Reductase Intermediate 1(ARI1) and Saccharomyces Aldehyde Reductase YGL039W genes, or (ii)Alcohol Dehydrogenase 7 (ADH7) and Genes de Respuesta a Estres 2 (GRE2)genes.
 2. The recombinant host cell of claim 1, wherein the recombinanthost cell further comprises a nucleic acid encoding an AROM polypeptide,a nucleic acid encoding a catechol-O-methyltransferase (COMT)polypeptide, a nucleic acid encoding a 3-dehydroshikimate dehydratase(3DSD) polypeptide, a nucleic acid encoding an aromatic carboxylic acidreductase (ACAR) polypeptide, a nucleic acid encoding aphosphopantetheine transferase (PPTase) polypeptide, a nucleic acidencoding an uridine 5′-diphosphoglucosyl transferase (UGT) polypeptideand/or a nucleic acid encoding a vanillyl alcohol oxidase (VAO).
 3. Therecombinant host cell of claim 1, wherein the recombinant host cell is amicroorganism.
 4. The recombinant host cell of claim 3, wherein themicroorganism is Saccharomyces cerevisiae, Schizosaccharomyces pombe orEscherichia coli.
 5. The recombinant host cell of claim 1, wherein therecombinant host cell is a plant cell.
 6. The recombinant host cell ofclaim 5, wherein the plant cell is a Physcomitrella cell or tobaccoplant cell.
 7. A recombinant yeast cell having the followingcharacteristics: (a) the recombinant yeast cell produces vanillin and/orvanillin glycoside; and (b) the recombinant yeast cell has a genedisruption or replacement of Alcohol Dehydrogenase 6 (ADH6) gene and agene disruption or replacement of: (i) Aldehyde Reductase Intermediate 1(ARI1) and Saccharomyces Aldehyde Reductase YGL039W genes, or (ii)Alcohol Dehydrogenase 7 (ADH7) and Genes de Respuesta a Estres 2 (GRE2)genes.
 8. The recombinant yeast cell of claim 7, wherein the recombinantyeast cell further comprises a nucleic acid encoding an AROMpolypeptide, a nucleic acid encoding a catechol-O-methyltransferase(COMT) polypeptide, a nucleic acid encoding a 3-dehydroshikimatedehydratase (3DSD) polypeptide, a nucleic acid encoding an aromaticcarboxylic acid reductase (ACAR) polypeptide, a nucleic acid encoding aphosphopantetheine transferase (PPTase) polypeptide, a nucleic acidencoding an uridine 5′-diphosphoglucosyl transferase (UGT) polypeptideand/or a nucleic acid encoding a vanillyl alcohol oxidase (VAO).
 9. Therecombinant yeast cell of claim 7, wherein the recombinant yeast cell isa member of the genus Saccharomyces.
 10. The recombinant yeast cell ofclaim 9, wherein the recombinant yeast cell is Saccharomyces cerevisiae.11. A method for producing vanillin and/or vanillin glycosidecomprising: (a) providing a recombinant host cell that produces vanillinand/or vanillin glycoside and has a gene disruption or replacement ofAlcohol Dehydrogenase 6 (ADH6) gene and a gene disruption or replacementof: (i) Aldehyde Reductase Intermediate 1 (ARM and SaccharomycesAldehyde, Reductase YGL039W genes, or (ii) Alcohol Dehydrogenase 7 (AMP)and Genes de Respuesta a Estres 2 (GRE2) genes; (b) cultivating saidrecombinant host cell for a time sufficient for said recombinant hostcell to produce vanillin and/or vanillin glycoside; and (c) isolatingvanillin and/or vanillin glycoside from said recombinant host cell orfrom the cultivation supernatant, thereby producing vanillin and/orvanillin glycoside.
 12. The method of claim 11, wherein the recombinanthost cell further comprises a nucleic acid encoding an AROM polypeptide,a nucleic acid encoding a catechol-O-methyltransferase (COMT)polypeptide, a nucleic acid encoding a 3-dehydroshikimate dehydratase(3DSD) polypeptide, a nucleic acid encoding an aromatic carboxylic acidreductase (ACAR) polypeptide, a nucleic acid encoding aphosphopantetheine transferase (PPTase) polypeptide, a nucleic acidencoding an uridine 5′-diphosphoglucosyl transferase (UGT) polypeptideand/or a nucleic acid encoding a vanillyl alcohol oxidase (VAO).
 13. Themethod of claim 11, wherein the recombinant host cell is amicroorganism.
 14. The method of claim 13, wherein the microorganism isSaccharomyces cerevisiae, Schizosaccharomyces pombe or Escherichia coli.15. The method of claim 11, wherein the recombinant host cell is a plantcell.
 16. The method of claim 15, wherein the plant cell is aPhyscomitrella cell or tobacco plant cell.